Recombinant DNA technology lect

Recombinant DNA Technology
Recombinant DNA technology procedures by
which DNA from different species can be isolated,
cut and spliced together -- new "recombinant "
molecules are then multiplied in quantity in
populations of rapidly dividing cells (e.g. bacteria,
yeast(.

The term gene cloning, recombinant DNA
technology and genetic engineering may seems
similar, however they are different techniques in
Biotechnology and they are interrelated

In the early 1970s it became possible to isolate a
specific piece of DNA out of the millions of base
pairs in a typical genome.

Currently it is relatively easy to cut out a specific
piece of DNA, produce a large number of copies ,
determine its nucleotide sequence, slightly alter it
and then as a final step transfer it back into cell in.

1.DNA molecules are digested with enzymes called
restriction endonucleases which reduces the size of
the fragments  Renders them more manageable
for cloning purposes

2.These products of digestion are inserted into a
DNA molecule called a vector  Enables desired
fragment to be replicated in cell culture to very high
levels in a given cell (copy(#

3.Introduction of recombinant DNA molecule into
an appropriate host cell
Transformation or transfection
Each cell receiving rDNA = CLONE
May have thousands of copies of rDNA molecules/cell after
DNA replication
As host cell divides, rDNA partitioned into daughter cells

4.Population of cells of a given clone is expanded,
and therefore so is the rDNA.
Amplification
DNA can be extracted, purified and used for molecular
analyses
Investigate organization of genes
Structure/function
Activation
Processing
Gene product encoded by that rDNA can be characterized
or modified through mutational experiments

Restriction Endonucleases
Endonuclease : Sequence specific nuclease that brak
the nucleic acid chains some where in the interior
rather than atb the ends of the molecules
Econuclease : Nuclease that remove the nucleotides
from the ends of the molecules

A. Origin and function
Bacterial origin = enzymes that cleave foreign DNA
Named after the organism from which they were
derived
EcoRI from Escherichia coli
BamHI from Bacillus amyloliquefaciens
Protect bacteria from bacteriophage infection
Restricts viral replication
Bacterium protects it’s own DNA by methylating
those specific sequence motifs

Over 200 enzymes identified, many available
commercially from biotechnology companies

B. Classes
Type I & III
-Cuts the DNA on both strands but at a non-specific location
at varying distances from the particular sequence that is
I : cleave the DNA at site located at 100 bp from the
recognition site
III : at about 24 bp
recognized by the restriction enzyme
-Therefore random/imprecise cuts
-Not very useful for rDNA applications

Type II
-Cuts both strands of DNA within the particular
sequence restriction site )) recognized by the restriction
enzyme
-Used widely for molecular biology procedures
-DNA sequence = symmetrical

Reads the same in the 5’ 3’ direction on both
strands = Palindromic Sequence
Some enzymes generate “blunt ends” (cut in
middle(
Others generate “sticky ends” (staggered cuts(
H-bonding possible with complementary tails
DNA ligase covalently links the two fragments together by
forming phosphodiester bonds of the phosphate-sugar
backbones

-EcoRI: Escherichia coli strain R, 1st
enzyme
-BamHI :Bacillus amyloliquefaciens strain H, 1st
enzyme
Diplococcus pneumoniae, 1st
enzyme- DpnI:
-HindIII :Haemophilus influenzae, strain D, 3rd
enzyme
-BglII :Bacillus globigii, 2nd
enzyme
-PstI :Providencia stuartii 164, 1st
enzyme
-Sau3AI :Staphylococcus aureus strain 3A, 1st
enzyme
-KpnI Klebsiella pneumoniae, 1st
enzyme

Restriction Enzymes
If two complementary strands of DNA are of equal
length, then they will terminate in a blunt end, as in
the following example:
55'-'-
CpTpGpApTpCpTpGpApCpTpGpApTpGpCpGpTpApTpGpCpTpApGpT-3-3''
33'-'-
GpApCpTpApGpApCpTpGpApCpTpApCpGpCpApTpApCpGpApTpCpA-5-5''

Restriction Enzymes
However, if one strand extends beyond the
complementary region, then the DNA is said to
possess an overhang:
55'-'-ApTpCpTpGpApCpT-3-3''
33'-'-TpApGpApCpTpGpApCpTpApCpG-5-5''

Restriction Enzymes
If another DNA fragment exists with a
complementary overhang, then these two overhangs
will tend to associate with each other and each strand
is said to possess a sticky end:

Restriction Enzymes
55'-'-ApTpCpTpGpApCpT pGpApTpGpCpGpTpApTpGpCpT--
33''
33'-'-TpApGpApCpTpGpApCpTpApCpGp CpApTpApCpGpA--
55''
Becomes
55'-'-ApTpCpTpGpApCpT pGpApTpGpCpGpTpApTpGpCpT-3-3''
33'-'-TpApGpApCpTpGpApCpTpApCpGp CpApTpApCpGpA-5-5''

Digestion of DNA by EcoRI to produce
cohesive ends ( Fig. 3.1(:

Creating recombinant DNA:
The first Recombinant DNA molecules were made
by Paul Berg at Stanford University in 1972.
In 1973 Herbert Boyer and Stanley Cohen created
the first recombinant DNA organisms.

Creating Recombinant DNA (Fig 3.2(:

Summary of Recombinant DNA technology
process:
Recombinant DNA technology requires DNA
extraction, purification, and fragmentation.
Fragmentation of DNA is done by specific
'restriction' enzymes and is followed by sorting and
isolation of fragments containing a particular gene.

Summary of Recombinant DNA technology
process:
This portion of the DNA is then coupled to a
carrier molecule.
The hybrid DNA is introduced into a chosen cell
for reproduction and synthesis.

Transformation and Antibiotic Selection
Transformation is the genetic alteration of a cell
resulting from the introduction, uptake and
expression of foreign DNA.

Transformation and Antibiotic Selection
There are more aggressive techniques for inserting
foreign DNA into eukaryotic cells. For example,
through electroporation.
Electroporation involves applying a brief
(milliseconds) pulse high voltage electricity to
create tiny holes in the bacterial cell wall that
allows DNA to enter.

Plasmids and Antibiotic resistance
Plasmids were discovered in the late sixties, and
it was quickly realized that they could be used to
amplify a gene of interest.
A plasmid containing resistance to an antibiotic
(usually ampicillin) or Tetracycline, is used as a
vector.

The gene of interest (resistant to Ampicillin) is
inserted into the vector plasmid and this newly
constructed plasmid is then put into E. coli that is
sensitive to ampicillin.( Text bk:Pg 58(
The bacteria are then spread over a plate that
contains ampicillin.

The ampicillin provides a selective pressure
because only bacteria that have acquired the
plasmid can grow on the plate.
Those bacteria which do not acquire the plasmid
with the inserted gene of interest will die.

As long as the bacteria grow in ampicillin, it will
need the plasmid to survive and it will continually
replicate it, along with the gene of interest that has
been inserted to the plasmid.

Fig 3.3 (a(.
Selecting a
Gene in a
plasmid and
Antibiotic
selection.

Human Gene cloning
Once inside a bacterium, the plasmid containing
the human cDNA can multiply to yield several
dozen replicas.

Reading materials:
Summary of Recombinant DNA and Cloning
(Fig. below(:
Isolation of two kinds of DNA
Treatment of plasmid and foreign DNA with the
same restriction enzyme
Mixture of foreign DNA with plasmids

Addition of DNA ligase
Introduction of recombinant plasmid into bacterial
cells
Production of multiple gene copies by gene cloning

Summary of Recombinant DNA and Cloning
(Fig.(:

This segment is "glued" into place using an enzyme
called DNA ligase.
The result is an edited, or recombinant, DNA
molecule.

Fig:
Inserting a
DNA
sample into
a Plasmid

Vectors for Gene Cloning
The choice of a vector depends on the design of the
experimental system and how the cloned gene will be
screened or utilized subsequently
Most vectors contain a prokaryotic origin of
replication allowing maintenance in bacterial cells.

Some vectors contain an additional eukaryotic origin
of replication allowing autonomous, episomal
replication in eukaryotic cells.
Multiple unique cloning sites are often included for
versatility and easier library construction.

-Antibiotic resistance genes and/or other selectable
markers enable identification of cells that have
acquired the vector construct.
-Some vectors contain inducible or tissue-specific
promoters permitting controlled expression of
introduced genes in transfected cells or transgenic
animals.

-Modern vectors contain multi-functional elements
designed to permit a combination of cloning, DNA
sequencing, in vitro mutagenesis and transcription
and episomal replication.

Main types of vectors
Plasmid, bacteriophage, cosmid, bacterial artificial
chromosome (BAC), yeast artificial chromosome
(YAC), yeast 2 micron plasmid, retrovirus,
baculovirus vector……

.Plasmid vector
-Covalently closed, circular, double stranded DNA
molecules that occur naturally and replicate
extrachromosomally in bacteria
-Many confer drug resistance to bacterial strains
-Origin of replication present (ORI(

-Bacterial cells may
contain
extrachromosomal DNA
called plasmids.
Plasmids are usually
represented by small,
circular DNA.
Some plasmids are
present in multiple
copies in the cell

Plasmid vectors are ≈1.2–3kb
and contain:
replication origin (ORI)
sequence
a gene that permits
selection,
Here the selective gene is
ampr; it encodes the enzyme
b-lactamase, which
inactivates ampicillin.
Exogenous DNA can be
inserted into the bracketed
region

Origin of
replication is a DNA
segment recognized by
the cellular DNA-
replication enzymes.
Without replication
origin, DNA cannot be
replicated in the cell

Many cloning vectors contain a
multiple cloning site or
polylinker: a DNA segment with
several unique sites for restriction
endonucleases located next to
each other
Restriction sites of the polylinker
are not present anywhere else in
the plasmid.
Cutting plasmids with one of the
restriction enzymes that recognize
a site in the polylinker does not
disrupt any of the essential
features of the vector

Examples
-pBR322
-One of the original plasmids used
-Two selectable markers (Amp and Tet resistance(
-Several unique restriction sites scattered throughout plasmid
(some lie within antibiotic resistance genes = means of screening
for inserts(
-ColE1 ORI

-pUC18
-Derivative of pBR322
-Advantages over pBR322:
-Smaller – so can accommodate larger DNA fragments
during cloning (5-10kbp(
-Higher copy # per cell (500 per cell = 5-10x more than
pBR322(
-Multiple cloning sites clustered in same location =
“polylinker”

LIGATION OF DNA SAMPLE AND
PLASMID DNA

TRANSFORMATION OF LIGATION PRODUCTS
The process of transferring exogenous DNA into
cells is call “transformation”
There are basically two general methods for
transforming bacteria. The first is a chemical
method utilizing CaCl2 and heat shock to promote
DNA entry into cells.
A second method is called electroporation based
on a short pulse of electric charge to facilitate DNA
uptake.

CHEMICAL TRANSFORMATION WITH CALCIUM
CHLORIDE

TRANSFORMATION BY
ELECTROPORATION

TERMS USED IN CLONING
DNA recombination.
The DNA fragment to be cloned is inserted into a
vector.
Transformation.
The recombinant DNA enters into the host cell
and proliferates.
Selective amplification.
A specific antibiotic is added to kill E. coli without
any protection. The transformed E. coli is
protected by the antibiotic-resistance gene
Isolation of desired DNA clones

CLONING VECTORS
Cloning vectors are DNA molecules that are used to
"transport" cloned sequences between biological
hosts and the test tube.
Cloning vectors share four common properties:
1.Ability to promote autonomous replication.
2. Contain a genetic marker (usually dominant) for
selection.
3. Unique restriction sites to facilitate cloning of insert
DNA.
4. Minimum amount of nonessential DNA to optimize
cloning.

Recombinant DNA technology lect

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Recombinant DNA technology lect